We propose to use the technique of cryoenzymology, i.e., subzero temperatures and fluid aqueous organic solvent systems, to provide a detailed, qualitative and quantitative understanding of the mechanisms of enzyme catalysis. Preliminary studies have shown the method to be well-suited for the detection and accumulation of intermediates in the enzyme-catalyzed reactions of specific substrates (both in the dissolved and crystalline states). By using appropriate spectral methods (e.g., fluorescence, Raman, e.s.r., nmr, c.d.) to monitor the reaction after initiation by mixing enzyme and substrate at a very low temperature, e.g., minus 100 degrees C, it will be possible to obtain kinetic, thermodynamic and some structural information about intermediates and their transformations. Further structural information will be obtained when possible from X-ray diffraction studies on the crystalline trapped intermediates. From the information obtained in this investigation we expect to be able to significantly contribute not only to the understanding of the mechanisms of action of these particular enzymes but also to further knowledge about the factors responsible for the efficiency of enzyme catalysis, and the events occurring during the dynamic processes of catalysis, in general. The low-temperature technique will also be used to detect and study intermediates in the folding and unfolding of ribonuclease. The following enzymes have been chosen as suitable representatives from a number of important classes of enzymes for study; lysozyme, ribonuclease, alcohol dehydrogenase, D-amino acid oxidase, glucose oxidase, and beta-glucosidase.